JPWO2021119402A5 - - Google Patents

Description

In another aspect, provided herein are devices for use in the methods provided herein. In some aspects, the device includes a light source and a sample holder.
[Invention 1001]
a. in the direction of 5' to 3',
i. optionally, a Unique Molecular Identifier (UMI) sequence;
ii. a first targeting domain; and
iii. First hybridization domain
a first nucleic acid comprising
b. in the direction of 5' to 3',
i. barcode domain; and
ii. A second hybridization domain that is substantially complementary to the first hybridization domain of the first nucleic acid
a second nucleic acid comprising
A barcode composition comprising
Said barcode composition, wherein at least one of the first or second hybridization domains comprises a photoreactive element.
[Invention 1002]
1002. The barcode composition of invention 1001, wherein said second nucleic acid further comprises a unique molecular identifier sequence at the 5' end.
[Invention 1003]
The barcode composition of invention 1001 or 1002, wherein said second nucleic acid further comprises a primer sequence at the 5' end.
[Invention 1004]
a. in the direction of 5' to 3',
i. optionally, a unique molecular identifier sequence;
ii. a first targeting domain; and
iii. First hybridization domain
a first nucleic acid comprising
b. in the direction of 5' to 3',
i. a second hybridization domain, the second hybridization domain being substantially complementary to the first hybridization domain of the first nucleic acid; and
ii. First barcode domain
a second nucleic acid comprising
A barcode composition comprising
Said barcode composition, wherein at least one of the first or second hybridization domains comprises a photoreactive element.
[Invention 1005]
The barcode of any of inventions 1001-1004, further comprising a third nucleic acid comprising a second barcode domain, wherein the second barcode domain is substantially complementary to the first barcode domain. Composition.
[Invention 1006]
1005. The barcode composition of invention 1005, wherein said third nucleic acid further comprises a unique molecular identifier sequence at the 5' end.
[Invention 1007]
The barcode composition of invention 1005 or 1006, wherein said third nucleic acid further comprises a primer sequence at the 5' end.
[Invention 1008]
a. in the direction of 5' to 3',
i. optionally, a unique molecular identifier sequence;
ii. a first targeting domain; and
iii. First hybridization domain
a first nucleic acid comprising
b. in the direction of 5' to 3',
i. a second hybridization domain, the second hybridization domain being substantially complementary to the first hybridization domain of the first nucleic acid; and
ii. a first barcode domain;
iii. Third hybridization domain
a second nucleic acid comprising
A barcode composition comprising
Said barcode composition, wherein at least one of the first or second hybridization domains comprises a photoreactive element and the third hybridization domain optionally comprises a photoreactive element.
[Invention 1009]
further comprising n additional nucleic acids;
n is an integer from 1 to 100;
each additional nucleic acid, in the 5' to 3' direction,
i. a first hybridization domain;
ii. barcode domain; and
iii. Second hybridization domain
including
the first hybridization domain of the nth nucleic acid is substantially complementary to the second hybridization domain of the (n-1)th nucleic acid;
the first hybridization domain of n=1 nucleic acids is substantially complementary to the third hybridization domain, and
1008. The barcode composition of the invention 1008, wherein at least one of the first or second hybridization domains of each nucleic acid comprises a photoreactive element.
[Invention 1010]
in the direction of 5' to 3',
i. a first cap hybridization domain that is substantially complementary to a second hybridization domain of the nth nucleic acid when n is 1 or greater, or n is 0; the cap hybridization domain, which is substantially complementary to the third hybridization domain when
ii. Second cap hybridization domain
a first capped nucleic acid strand comprising
further comprising
The barcode composition of invention 1008 or 1009, wherein the first cap hybridization domain optionally comprises a photoreactive element.
[Invention 1011]
in the direction of 5' to 3',
i. a primer sequence domain;
ii. optionally a unique molecular identifier (UMI) sequence; and
iii. a hybridization domain that is substantially complementary to the second cap hybridization domain of the first cap nucleic acid
a second capped nucleic acid strand comprising
further comprising
The barcode composition of invention 1010, wherein at least one of the second cap hybridization domain of the first cap nucleic acid strand and the hybridization domain of the second nucleic acid strand comprises a photoreactive element.
[Invention 1012]
1012. The barcode composition of any of inventions 1001-1011, wherein said first nucleic acid is RNA or an RNA transcript and optionally the first hybridization domain comprises a poly(A) sequence.
[Invention 1013]
1013. The barcode composition of any of inventions 1001-1012, wherein said first nucleic acid further comprises a primer sequence at the 5' end.
[Invention 1014]
1013. The barcode composition of any of inventions 1001-1013, wherein the first targeting domain of said first nucleic acid is substantially complementary to the target nucleic acid.
[Invention 1015]
Either the target nucleic acid is conjugated to a target binding agent, or the target nucleic acid is conjugated to a target molecule, or the target nucleic acid is contained within a target molecule (such as RNA), or the target Either the nucleic acid is expressed by the target cell, or the target nucleic acid is directly on the target molecule or cell, or by chemical cross-linking, gene encoding, viral transduction, transfection, conjugation, cell fusion, cell A barcode composition of the invention 1014 that is indirectly presented via internalization, hybridization, a DNA binding protein or adapter molecule, such as a target binding ligand.
[Invention 1016]
Amino acids, peptides, proteins, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, lipopolysaccharides, lectins, nucleosides, nucleotides, nucleic acids, vitamins, steroids, hormones, cofactors, receptors and receptors The barcode composition of the invention 1015 selected from the group consisting of ligands, optionally wherein the target binding agent is an antibody or antigen binding fragment thereof.
[Invention 1017]
1016. The barcode composition of any of Inventions 1001-1016, wherein each domain independently comprises a 1-letter code, a 2-letter code, a 3-letter code, or a 4-letter code.
[Invention 1018]
The barcode composition of any of Inventions 1001-1017, wherein each domain independently comprises zero or at least one nucleic acid modification.
[Invention 1019]
The barcode composition of the invention 1018, wherein the nucleic acid modifications are selected from the group consisting of nucleobase modifications, sugar modifications, and internucleotide linkage modifications.
[Invention 1020]
The barcode composition of any of Inventions 1001-1019, wherein each domain is independently 1-1000 nucleotides in length.
[Invention 1021]
1021. The barcode composition of any of the inventions 1001-1020, wherein the UMI of a nucleic acid is incorporated into one of the other domains of the same nucleic acid.
[Invention 1022]
The barcode composition of any of inventions 1001-1021, wherein at least one of the nucleic acids comprises a cleavable spacer.
[Invention 1023]
The barcode composition of invention 1022, wherein the cleavable spacer is a photocleavable spacer.
[Invention 1024]
The barcode composition of any of inventions 1001-1023, further comprising a detectable label.
[Invention 1025]
The barcode composition of the invention 1024, wherein a detectable label is contained within one of said nucleic acids.
[Invention 1026]
Detectable labels include fluorescent molecules, nanoparticles, stable isotopes, radioisotopes, nucleotide chromophores, enzymes, enzyme substrates, chemiluminescent and bioluminescent moieties, echogenic substances, non-metallic isotopes, optical reporters, common The barcode composition of the invention 1024 or 1025 selected from the group consisting of magnetic metal ions and ferromagnetic metals and optionally the detectable label is a fluorophore.
[Invention 1027]
The barcode composition of any of inventions 1001-1026, further comprising a polymerase.
[Invention 1028]
The barcode composition of the invention 1027, wherein the polymerase is a strand displacement polymerase.
[Invention 1029]
The barcode composition of any of inventions 1001-1028, further comprising buffers or salts for nucleic acid synthesis.
[Invention 1030]
The barcode composition of any of inventions 1001-1029, further comprising natural or synthetic nucleotide triphosphates or deoxynucleotide triphosphates.
[Invention 1031]
The barcode composition of any of inventions 1001-1030, further comprising a targeting element.
[Invention 1032]
The barcode composition of the invention 1031, wherein the target elements are immobilized on the substrate surface.
[Invention 1033]
A barcode composition of the invention 1032, wherein the target elements are fixed in a predetermined pattern on the substrate surface.
[Invention 1034]
The barcode composition of any of inventions 1031-1033, wherein the target element is a nucleic acid, lipid, sugar, small molecule, microorganism or fragment thereof, polypeptide, and/or biological material.
[Invention 1035]
The barcode composition of the invention 1034, wherein the biological material is selected from the group consisting of tissue, cells, organoids, engineered tissue; and extracellular matrix.
[Invention 1036]
Substrate is glass, transparent polymer, polystyrene, hydrogel, metal, ceramic, paper, agarose, gelatin, alginate, dextran, iron oxide, stainless steel, gold, copper, silver chloride, polycarbonate, polydimethylsiloxane, polyethylene, acrylonitrile The barcode composition of any of Inventions 1031-1035, selected from the group consisting of butadiene styrene, cycloolefin polymers, cycloolefin copolymers, streptavidin, resins, and biomaterials.
[Invention 1037]
The barcode composition of any of inventions 1001-1036, wherein the photoreactive elements are photoreactive nucleotides, optionally the photoreactive nucleotides are CNVK or CNVD bridging bases.
[Invention 1038]
The barcode composition of any of inventions 1001-1037, further comprising PCR primers.
[Invention 1039]
The barcode composition of any of inventions 1001-1038, further comprising a light source, optionally the light source is a UV light source.
[Invention 1040]
A barcode composition of any of the inventions 1001-1039 in kit form.
[Invention 1041]
A method of detecting a target mRNA, the method comprising the steps of:
a. Hybridizing the target mRNA (first nucleic acid) and the second nucleic acid,
i. said mRNA comprises a first hybridization domain comprising a poly A sequence;
ii. the second nucleic acid, in the 5′ to 3′ direction,
1. a second hybridization domain, the second hybridization domain being substantially complementary to the first hybridization domain and comprising a photoreactive element; and
2. First barcode domain
stages, including
b. photocrosslinking the mRNA and a second nucleic acid, thereby forming a probe-primer complex;
c. synthesizing a recording nucleic acid from the probe-primer complex; and
d. Detecting the recorded nucleic acid.
[Invention 1042]
A method of detecting a target nucleic acid, the method comprising the steps of:
a. hybridizing the target nucleic acid with the first nucleic acid and hybridizing the second nucleic acid with the first nucleic acid, comprising:
i. the first nucleic acid, in the 5′ to 3′ direction,
1. optionally, a Unique Molecular Identifier (UMI) sequence;
2. a first targeting domain substantially complementary to the nucleic acid of the target element; and
3. First hybridization domain
includes;
ii. the second nucleic acid, in the 5′ to 3′ direction,
1. a second hybridization domain, the second hybridization domain being substantially complementary to the first hybridization domain; and
2. First barcode domain
including
at least one of the first or second hybridization domains comprises a photoreactive element;
b. photocrosslinking the first nucleic acid and the second nucleic acid, thereby forming a probe-primer complex;
c. optionally, denaturing the probe-primer complex from the target nucleic acid;
d. synthesizing a recording nucleic acid from the probe-primer complex; and
e. Detecting the recorded nucleic acid.
[Invention 1043]
The method of invention 1041 or 1042, wherein said second nucleic acid further comprises a unique molecular identifier (UMI) sequence at its 5' end.
[Invention 1044]
The method of any of inventions 1041-1043, wherein said second nucleic acid further comprises a primer sequence at the 5' end.
[Invention 1045]
Inventions 1041-1044, wherein said detecting comprises sequencing of recorded nucleic acids, light microscopy, high throughput scanners, confocal microscopy, light sheet microscopy, electron microscopy, atomic force microscopy, or the naked eye. Either method.
[Invention 1046]
The method of the invention 1045, further comprising cutting, uncrosslinking, removing or reversing photocrosslinks and amplifying the recorded nucleic acid prior to sequencing.
[Invention 1047]
1046. The method of the invention 1046, wherein said cleaving, de-crosslinking, removing or reversing uses light of wavelength 300-350 nm, optionally 312 nm.
[Invention 1048]
A method of detecting a target mRNA, the method comprising the steps of:
a. Hybridizing the target mRNA (first nucleic acid) and the second nucleic acid,
i. said mRNA comprises a first hybridization domain comprising a poly A sequence;
ii. the second nucleic acid, in the 5′ to 3′ direction,
1. a second hybridization domain substantially complementary to the first hybridization domain of said mRNA and comprising a photoreactive element; and
2. First barcode domain
stages, including
b. photocrosslinking the mRNA and a second nucleic acid, thereby forming a first complex;
c. hybridizing a third nucleic acid to the second nucleic acid in the first complex, thereby forming a probe-primer complex, wherein the third nucleic acid is the first comprising a second barcode domain substantially complementary to the barcode domain;
d. synthesizing a recording nucleic acid from the probe-primer complex; and
e. Detecting the recorded nucleic acid.
[Invention 1049]
A method of detecting a target nucleic acid, the method comprising the steps of:
a. hybridizing a target nucleic acid with a first nucleic acid and hybridizing a second nucleic acid to the first nucleic acid, comprising:
i. the first nucleic acid, in the 5′ to 3′ direction,
1. optionally, a Unique Molecular Identifier (UMI) sequence;
2. a first targeting domain, the first targeting domain being substantially complementary to the target nucleic acid; and
3. First hybridization domain
includes;
ii. the second nucleic acid, in the 5′ to 3′ direction,
1. a second hybridization domain, the second hybridization domain being substantially complementary to the first hybridization domain of the first nucleic acid;
2. First barcode domain
including
at least one of the first or second hybridization domains comprises a photoreactive element;
b. photocrosslinking the first nucleic acid and the second nucleic acid, thereby forming a first complex;
c. optionally, denaturing the first complex from the target nucleic acid;
d. hybridizing a third nucleic acid to the second nucleic acid in the first complex, thereby forming a probe-primer complex, wherein the third nucleic acid is the first comprising a second barcode domain substantially complementary to the barcode domain;
e. synthesizing a recording nucleic acid from the probe-primer complex; and
f. Detecting the recorded nucleic acid.
[Invention 1050]
The method of invention 1048 or 1049, wherein said third nucleic acid further comprises a unique molecular identifier (UMI) sequence at its 5' end.
[Invention 1051]
The method of any of inventions 1048-1050, wherein said third nucleic acid further comprises a primer sequence at the 5' end.
[Invention 1052]
Invention 1048-1051, wherein said detecting comprises sequencing of recorded nucleic acids, light microscopy, high throughput scanners, confocal microscopy, light sheet microscopy, electron microscopy, atomic force microscopy, or the naked eye. Either method.
[Invention 1053]
The method of invention 1052, further comprising amplifying the recorded nucleic acid prior to sequencing.
[Invention 1054]
A method of detecting a target nucleic acid, the method comprising the steps of:
a. hybridizing the target nucleic acid and the first nucleic acid, comprising:
i. the first nucleic acid, in the 5′ to 3′ direction,
1. optionally, a Unique Molecular Identifier (UMI) sequence;
2. a first targeting domain, the first targeting domain being substantially complementary to the target nucleic acid; and
3. First hybridization domain
stages, including
b. preparing a concatemer by hybridizing n additional nucleic acids and photocrosslinking the additional nucleic acids with the first complex, wherein n is an integer from 1 to 100; the additional nucleic acid, in the 5' to 3' direction,
i. a first hybridization domain;
ii. barcode domain; and
iii. Second hybridization domain
including
The first hybridization domain of the nth nucleic acid is substantially complementary to the second hybridization domain of the (n−1)th nucleic acid, and the first hybridization domain of n=1 nucleic acid is is substantially complementary to the first hybridization domain of the first nucleic acid, and at least one of the first or second hybridization domains of each nucleic acid comprises a photoreactive element;
c. hybridizing the first capped nucleic acid strand and the concatemer, thereby forming a capped concatemer, wherein the first capped nucleic acid comprises
i. a first cap hybridization domain, the first cap hybridization domain being substantially complementary to the second hybridization domain of the nth nucleic acid; and
ii. Second cap hybridization domain
stages, including
d. hybridizing a second capped nucleic acid strand to the capped concatemer, thereby forming a concatemer-primer complex, wherein the second capped nucleic acid strand, in the 5′ to 3′ direction,
i. a primer sequence domain;
ii. optionally a unique molecular identifier (UMI) sequence; and
iii. A hybridization domain that is substantially complementary to the second cap hybridization domain of the first cap nucleic acid
a stage comprising;
e. Detecting a concatemer-primer complex, or synthesizing a recording nucleic acid from the concatemer-primer complex and detecting the recording nucleic acid.
[Invention 1055]
The method of Invention 1054, wherein said detecting step comprises sequencing of recorded nucleic acids, light microscopy, high throughput scanners, confocal microscopy, light sheet microscopy, electron microscopy, atomic force microscopy, or the naked eye. .
[Invention 1056]
The method of the invention 1055, further comprising amplifying the recorded nucleic acid prior to sequencing.
[Invention 1057]
The method of any of inventions 1041-1054, wherein said photocrosslinking is performed in an aqueous solution.
[Invention 1058]
1055. The method of any of inventions 1041-1055, wherein said photocrosslinking uses light of wavelength 350-400 nm, optionally 365 nm.
[Invention 1059]
The method of any of inventions 1041-1058, further comprising one or more washing steps.
[Invention 1060]
The method of any of inventions 1041-1059, wherein the target nucleic acid is conjugated with a target binding ligand.
[Invention 1061]
Target binding ligands are amino acids, peptides, proteins, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, lipopolysaccharides, lectins, nucleosides, nucleotides, nucleic acids, vitamins, steroids, hormones, cofactors, receptors and receptors A method of the invention 1060, wherein the target binding ligand is selected from the group consisting of ligands and optionally the target binding ligand is an antibody or antigen binding fragment thereof.
[Invention 1062]
The method of any of inventions 1041-1061, wherein the target nucleic acid is contained in a biological material.
[Invention 1063]
The method of invention 1062, wherein the biological material is selected from the group consisting of tissue, cells, organoids, artificial tissue, and extracellular matrix.
[Invention 1064]
The method of any of Inventions 1041-1063, wherein the target nucleic acid is immobilized on a substrate surface.
[Invention 1065]
The method of any of Inventions 1041-1064, wherein the target nucleic acid is immobilized on the substrate surface in a predetermined pattern.
[Invention 1066]
Substrate is glass, transparent polymer, polystyrene, hydrogel, metal, ceramic, paper, agarose, gelatin, alginate, dextran, iron oxide, stainless steel, gold, copper, silver chloride, polycarbonate, polydimethylsiloxane, polyethylene, acrylonitrile The method of invention 1065 selected from the group consisting of butadiene styrene, cycloolefin polymers, cycloolefin copolymers, streptavidin, resins, and biomaterials.
[Invention 1067]
The method of any of inventions 1041-1066, wherein said first nucleic acid further comprises a primer sequence at the 5' end.
[Invention 1068]
1067. The method of any of the inventions 1041-1067, wherein each domain independently comprises a 1-letter code, a 2-letter code, a 3-letter code, or a 4-letter code.
[Invention 1069]
The method of any of Inventions 1041-1068, wherein each domain independently comprises zero or at least one nucleic acid modification.
[Invention 1070]
The method of the invention 1069, wherein the nucleic acid modifications are selected from the group consisting of nucleobase modifications, sugar modifications, and internucleotide linkage modifications.
[Invention 1071]
The method of any of Inventions 1041-1070, wherein each domain is independently 1-1000 nucleotides in length.
[Invention 1072]
The method of any of inventions 1041-1071, wherein the UMI of a nucleic acid is incorporated into the barcode domain or probe domain of the same nucleic acid.
[Invention 1073]
The method of any of inventions 1041-1072, wherein at least one of the nucleic acids comprises a cleavable spacer.
[Invention 1074]
The method of Invention 1073, wherein the cleavable spacer is a photocleavable spacer.
[Invention 1075]
The method of any of inventions 1041-1074, wherein at least one of the nucleic acids comprises a detectable label.
[Invention 1076]
Detectable labels include fluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, enzyme substrates, chemiluminescent and bioluminescent moieties, echogenic substances, non-metallic isotopes, optical reporters, paramagnetic metal ions, and ferromagnetic 1075. The method of the invention 1075, wherein the detectable label is selected from the group consisting of metals and optionally the detectable label is a fluorophore.
[Invention 1077]
1076. The method of any of inventions 1041-1076, wherein synthesizing said recording nucleic acid comprises using a strand displacement polymerase.
[Invention 1078]
The method of any of inventions 1041-1077, further comprising selecting one or more specific regions of interest for irradiation or detection.
[Invention 1079]
The method of invention 1078, wherein the step of selecting one or more particular regions is manual or computer assisted.
[Invention 1080]
The method of invention 1078 or 1079, wherein said selection is based on one or more phenotypic markers.
[Invention 1081]
The method of invention 1080, wherein the one or more phenotypic markers is fluorescence, shape, intensity, tissue staining, antibody staining, or morphology.
[Invention 1082]
The method of any of inventions 1041-1081, further comprising software to automatically detect one or more regions of interest for spatial illumination or detection.
[Invention 1083]
A method for linearly, combinatorially or spatially barcoding multiple targets in a sample, the method comprising the steps of:
a. hybridizing a target nucleic acid strand in each member of the plurality of targets with a first nucleic acid strand, wherein the target nucleic acid strand is different in each member of the plurality of targets and the target nucleic acid strand is a different nucleic acid; either contained within a molecule, or the target nucleic acid strand is conjugated to one member of a plurality of targets, or the target nucleic acid strand is expressed by the cell, or the target nucleic acid strand is or directly onto cells, or indirectly via chemical cross-linking, gene encoding, viral transduction, transfection, conjugation, cell fusion, intracellular uptake, hybridization, DNA binding proteins or target binders/ligands is effectively presented, and
i. the first nucleic acid strand, in the 5′ to 3′ direction,
1. optionally, a Unique Molecular Identifier (UMI) sequence;
2. a first targeting domain, the first targeting domain being substantially complementary to the target nucleic acid; and
3. First hybridization domain
stages, including
b. preparing a concatemer by hybridizing one or more additional nucleic acid strands in a stepwise fashion and photocrosslinking the additional nucleic acid strands with the first complex, said photocrosslinking selecting a predetermined region of the sample and exposing the predetermined region to light after hybridizing each additional nucleic acid strand, thereby cross-linking complementary hybridization domains; removing any uncrosslinked additional nucleic acid strand after exposure and prior to hybridization of the next additional nucleic acid strand;
each additional nucleic acid strand, in the 5' to 3' direction,
i. a first hybridization domain;
ii. barcode domain; and
iii. Second hybridization domain
including
the first hybridization domain of the nth additional nucleic acid strand is substantially complementary to the second hybridization domain of the (n−1)th additional nucleic acid strand, and the first additional nucleic acid strand is substantially complementary to the first hybridization domain of the first nucleic acid strand, and at least one of the first or second hybridization domains of each nucleic acid strand has a step comprising a photoreactive element; and
c. detecting the concatemers and/or synthesizing the recording nucleic acids from the concatemers and detecting the recording nucleic acids;
[Invention 1084]
The method of invention 1083, wherein at least one member of the plurality of targets is contained within another nucleic acid molecule.
[Invention 1085]
The present invention 1083 wherein at least one member of the plurality of targets is contained within another nucleic acid molecule independently selected from the group consisting of RNA, RNA transcripts, genomic DNA, nucleic acid amplification products, and any combination thereof. Or 1084 ways.
[Invention 1086]
The method of any of inventions 1083-1085, wherein at least one member of the plurality of targets is a cDNA.
[Invention 1087]
The method of any of inventions 1083-1086, wherein at least one member of the plurality of targets is a non-nucleic acid molecule conjugated to the target nucleic acid strand.
[Invention 1088]
The method of any of inventions 1083-1087, wherein at least one member of the plurality of targets is a non-nucleic acid molecule conjugated to the target nucleic acid strand via a targeting binding agent linked to the target nucleic acid strand.
[Invention 1089]
Amino acids, peptides, proteins, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, lipopolysaccharides, lectins, nucleosides, nucleotides, nucleic acids, vitamins, steroids, hormones, cofactors, receptors and The method of any of Inventions 1083-1088, wherein the target binding agent is selected from the group consisting of receptor ligands and optionally the target binding agent is an antibody or antigen binding fragment thereof.
[Invention 1090]
The method of any of inventions 1083-1089, wherein at least one member of the plurality of targets is a nucleic acid and at least one member of the plurality of targets is a non-nucleic acid molecule.
[Invention 1091]
The method of any of inventions 1083-1090, wherein at least one member of the plurality of targets is a protein.
[Invention 1092]
The method of any of inventions 1083-1091, wherein the sample is biological material.
[Invention 1093]
The method of any of inventions 1083-1092, wherein the sample is a biological material selected from the group consisting of tissue, cells, organoids, artificial tissue, and extracellular matrix.
[Invention 1094]
The method of any of inventions 1083-1092, wherein the sample is selected from the group consisting of whole tissue, tissue area, cell collection, single cell, subcellular area, and any combination thereof.
[Invention 1095]
The method of any of inventions 1083-1094, wherein the photoreactive element is CNVK.
[Invention 1096]
The method of any of Inventions 1083-1095, wherein the photoreactive element inhibits or blocks the activity of a polymerase, optionally the polymerase is a strand displacement polymerase.
[Invention 1097]
1096. The method of any of the inventions 1083-1096, comprising detecting concatemers and/or recording strands by imaging methods and sequencing of the recording nucleic acids for multimodal integrated analysis of predetermined regions of the sample.
[Invention 1098]
detecting concatemers and/or recording strands by imaging methods and sequencing of the recorded nucleic acids to correlate sequences of the recorded strands to spatial locations for multimodal integrated analysis of a given region of the sample. Any method from ~1097.
[Invention 1099]
Invention 1083-1098, wherein said detecting comprises sequencing of recorded nucleic acids, light microscopy, high throughput scanners, confocal microscopy, light sheet microscopy, electron microscopy, atomic force microscopy, or the naked eye. either way.
[Invention 1100]
1099. The method of the invention 1099, further comprising amplifying the recorded nucleic acid prior to sequencing.
[Invention 1101]
The method of the invention 1100, further comprising cutting, uncrosslinking, removing or reversing photocrosslinks and amplifying the recorded nucleic acid prior to sequencing.
[Invention 1102]
The method of any of inventions 1083-1101, wherein said photocrosslinking uses light of wavelength 350-400 nm, optionally 365 nm.
[Invention 1103]
1103. The method of any of inventions 1083-1102, wherein each domain independently comprises a 1-letter code, a 2-letter code, a 3-letter code, or a 4-letter code.
[Invention 1104]
The method of any of inventions 1083-1103, wherein at least one of the nucleic acid strands comprises a detectable label.
[Invention 1105]
Detectable labels include fluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, enzyme substrates, chemiluminescent and bioluminescent moieties, echogenic substances, non-metallic isotopes, optical reporters, paramagnetic metal ions, and ferromagnetic The method of invention 1104, wherein the detectable label is selected from the group consisting of metals and optionally the detectable label is a fluorophore.
[Invention 1106]
The method of any of inventions 1083-1105, wherein synthesizing said recording nucleic acid comprises using a strand displacement polymerase.
[Invention 1107]
1107. The method of any of inventions 1083-1106, wherein selecting said predetermined area is manual or computer assisted.
[Invention 1108]
Use of the method of any of inventions 1040-1107 to screen a library of candidates for treatment by imaging and barcoding a region of interest by any of the methods of invention 1040-1107 Said use comprising identifying one or more phenotypic markers.
[Invention 1109]
Use of the invention 1108, wherein the one or more phenotypic markers are fluorescence, shape, intensity, tissue staining, antibody staining, or morphology.
[Invention 1110]
identification of candidates for screening, identification of drug targets, identification of biomarkers, profiling, characterization of phenotypic vs. genotypic cell state, generation of new disease models, characterization of cell and disease models, differentiation state and Cell state characterization, tissue mapping, multidimensional analysis, high-content screening, machine learning-based clustering or classification, cell therapy development, CAR-T therapy development, antibody screening, personalized medicine, cell enrichment, and any of these Use of the method of any of the inventions 1040-1107 for combination.
[Invention 1111]
Invention 1108- wherein the candidate is selected from the group consisting of small molecule drugs, biologics, therapeutic nucleic acids, gene or cell therapies, siRNAs, gRNAs, peptides, proteins, antibodies, metabolites, hormones, and DNA-encoding libraries Any use of 1110.
[Invention 1112]
A kit of invention 1040 for use in a method for in vitro, in vivo, in situ or intoto barcoding of biomolecules using any of the methods of invention 1083-1111.

In some embodiments, the composition further comprises a first cap nucleic acid strand and a second cap nucleic acid strand, wherein the second cap nucleic acid strand extends, in a 5' to 3' direction, the primer sequence domain; a unique molecular identifier sequence; and a hybridization domain, wherein the hybridization domain is substantially complementary to the second cap hybridization domain of the first cap nucleic acid, and of the first cap nucleic acid strand At least one of the second cap hybridization domain and the second cap nucleic acid hybridization domain comprises a photoreactive element.

In yet another aspect, provided herein are methods for detecting a target nucleic acid. Generally, the method includes preparing concatemers. For example, the method comprises (i) hybridizing a target nucleic acid and a first nucleic acid, wherein the first nucleic acid comprises, in the 5′ to 3′ direction, any unique identifier sequence, a targeting domain and comprising a hybridization domain, wherein the first targeting domain is substantially complementary to the target nucleic acid; (ii) hybridizing n additional nucleic acids, e.g. wherein n is an integer from 1 to 100 and each additional nucleic acid is comprising a first hybridization domain, a barcode domain and a second hybridization domain, wherein the first hybridization domain of the nth nucleic acid is substantially the second hybridization domain of the (n−1)th nucleic acid substantially complementary, the first hybridization domain of n=1 nucleic acids being substantially complementary to the hybridization domain of the first nucleic acid, the first or second hybridization domain of each nucleic acid comprises a photoreactive element, and at least one of the n=1 nucleic acid first hybridization domain and the first nucleic acid hybridization domain comprises a photoreactive element; iii) hybridizing the first cap nucleic acid strand with the concatemer, thereby forming a capped concatemer, wherein the first cap nucleic acid comprises the first cap hybridization domain and the second cap hybridization domain; (iv) a concatemer capped with a second cap nucleic acid strand comprising a hybridization domain, wherein the first cap hybridization domain is substantially complementary to the second hybridization domain of the nth nucleic acid; to thereby form a concatemer-primer complex, wherein the second cap nucleic acid strand is, in the 5′ to 3′ direction, the primer sequence domain, the optional unique molecular identifier sequence and the hybridization wherein the hybridization domain of the second cap nucleic acid is substantially complementary to the second cap hybridization domain of the first cap nucleic acid, and the hybridization domain of the second cap nucleic acid and the second key of the first capped nucleic acid at least one of the cap hybridization domains comprises a photoreactive element; (v) detecting the concatemer-primer complex, or synthesizing the recording nucleic acid from the concatemer-primer complex and detecting the recording nucleic acid. including.

Figures 1A-1C show dual light induced barcoding (strategy 1). FIG. 1A shows that probe sequences bind to targets of interest followed by barcode-containing primers. Upon irradiation with UV light of the appropriate wavelength, the primers become covalently linked (cross-linked) to the probe sequence, and a polymerase is used to copy the complete recording strand followed by cross-linking with a different wavelength of light. reverse. Recorded amplicons may first be PCR amplified before being sent for sequencing. FIG. 1B shows that the probe sequence can bind in situ to any nucleic acid-labeled entity in addition to the genomic/transcriptome target, such as a DNA-conjugated antibody that binds to the target protein. . FIG. 1C shows that a non-targeted approach can also be used for barcoding. For example, the polyA tail of an mRNA transcript can be attached to a barcode primer, which can then be crosslinked as previously described. Reverse transcription is used to copy part or all of the mRNA transcript sequence prior to subsequent preparation steps and sequencing. See description of Figure 1A-1. See description of Figure 1A-1. See description of Figure 1A-1. See description of Figure 1A-1. Figures 2A-2D show light induced barcoding (strategy 2) using a barcoded bridge arrangement. Figure 2A shows that the probe sequence binds to the target of interest followed by a barcode-containing bridge strand. Upon irradiation with an appropriate wavelength of UV light, the bridge becomes covalently linked (cross-linked) to the probe sequence and the probe-bridge complex is formed before the corresponding primer hybridizes to the barcode sequence. can be denatured into A polymerase is used to copy the complete recording strand, which can then be PCR amplified prior to sequencing. If a strand-displacing polymerase is used, the polymerization reaction can occur even when the probe remains bound to the target (part (Fig. 2B)). FIG. 2C shows that a non-targeted approach can also be used for barcoding. For example, the polyA tail of an mRNA transcript may be attached to a barcode bridge containing several T bases. FIG. 2D shows that these barcode bridges can then be crosslinked and prepared as described above for sequencing (eg, by reverse transcription). Sequencing is then used to recover the transcript plus barcode information. See description of Figure 2A-1. See description of Figure 2A-1. See description of Figure 2A-1. See description of Figure 2A-1. See description of Figure 2A-1. Figures 3A-3C show light-induced barcoding (strategy 3) using concatemer assembly. Figure 3A shows that the probe sequence binds to the target of interest followed by the barcode strand. Upon irradiation with UV light of the appropriate wavelength, the barcode becomes covalently linked (crosslinked) to the probe sequence. Concatemers are formed through repeated barcode hybridization and cross-linking reactions. FIG. 3B shows that a strand displacement polymerase can be used to copy the complete recording strand through a cross-junction synthesis reaction, which can then be PCR amplified prior to sequencing. The sequence reveals the integrated information of the barcode sequence and the target sequence. Concatemer assemblies may first be denatured from the sample/surface prior to priming and cross-junction synthesis (part (FIG. 3C)). See description of Figure 3A-1. See description of Figure 3A-1. See description of Figure 3A-1. Figures 4A-4D show light induced barcoding. FIG. 4A shows that a basic sequence-specific cross-linking reaction requires two complementary or near-complementary sequences, one containing a CNVK modification, that bind to each other. Exposure to UV light causes covalent linking (crosslinking) of the chains. FIG. 4B shows that cross-linking can also be localized to a specific region or set of regions (using strategy 1 chemistry as described above) by localized irradiation to those regions. For example, a probe sequence containing CNVK binds but is crosslinked only in some regions, and then washing away all uncrosslinked strands results in probes that bind only in the irradiated regions. FIG. 4C shows that repeated hybridizations using barcode primers with different barcode sequences (eg, B1-Bn), spatial patterning cross-linking and washing rounds can be used to label distinct regions. show. After sequencing (which can be done with all recordings synthesized simultaneously and pooled during sequencing), the barcode sequence and target/transcript integration information is recovered. Also, repeated spatial patterning cross-linking can be performed similar to the second barcode chemistry (strategy 2) previously described, but with different rounds of binding barcode bridge strands rather than different barcode primers. (Part (Fig. 4D)). See description of Figure 4A. See description of Figure 4A. See description of Figure 4A. Figures 5A-5C show light-induced combinatorial barcoding. FIG. 5A Combinatorial light-guided barcode assembly is achieved via repeated hybridization of barcode strands with different barcode sequences (e.g., sequences 0 and 1), spatial patterning cross-linking, and washing rounds. indicates that FIG. 5B shows that each individual region can undergo a unique assembly order (e.g., 1010010 or 0011101 in the example shown), or multiple regions can undergo the same assembly sequence if desired. . FIG. 5C shows that the sequence of assembled barcode sequence plus original probe sequence information is synthesized in the recording strand through the cross-junction synthesis reaction. PCR amplification may be performed prior to sequencing the recording. See description of Figure 5A. See description of Figure 5A. Figures 6A-6F demonstrate experimental verification of spatial patterning bridges. FIG. 6A shows the use of CNVK (grey closed circles) modified barcode strands in conjunction with a spatial photomask to direct the cross-linking of barcodes to RNA targets in cell collections. The barcode strand contains both the barcode sequence (blue and purple) and the Cy3b fluorophore (green star). Repeated light-induced barcode construction can proceed through successive washing and UV cross-linking events. FIG. 6B shows the final cross-linking step, demonstrating delivery and cross-linking of strands carrying primer binding sites (orange) to Cy5-labeled primer strands (orange strands with magenta stars). Full area cross-linking was performed for this step. Figure 6C shows EY.T4 cells labeled with DAPI (blue channel). No cross-linking. FIG. 6D shows that a spatial mask was applied to crosslink cellular ribosomal RNA with Cy3b (green channel) labeled barcode strands. The green channel illustrates successful cross-linking in a cross-rectangular pattern after formamide wash. Figure 6E shows a closer view of panel (d) at the "intersection" point between the two rectangles. Figure 6F shows imaging in the DAPI (blue), Cy3b (green) and Cy5 (magenta) channels after the final primer capping set shown in panel (Figure 6B). Cy5-labeled strands are expected to cross-link all cells due to global UV cross-linking. Cells containing both the barcoded and primer strands are expected to overlap in both the green and magenta channels and appear white in the channel overlay. Note, magenta channel contrast was scaled to match bar-coded cells expected to have a 3-fold higher Cy3b fluorophore compared to Cy5. See description of Figure 6A. See description of Figure 6A. See description of Figure 6A. See description of Figure 6A. See description of Figure 6A. Figures 7A-7C show the repeated assembly of up to three junctions of concatemers. FIG. 7A shows a schematic representation of repetitive junction assembly using Cy3b-labeled barcode strands and Cy5-labeled primers. Figures 7A-7C show the repeated assembly of up to three junctions of concatemers. FIG. 7B shows a representative schematic of cross-junction synthesis of one-junction and three-junction assemblies followed by PCR amplification of recordings. Figures 7A-7C show the repeated assembly of up to three junctions of concatemers. FIG. 7C shows a PAGE denaturing gel showing PCR products and no probe controls for two experiments. Figures 8A-8C show experimental validation of cellular level spatial labeling. Figure 8A shows a mixture of cells exhibiting different phenotypic markers. GFP-transfected cells (green circles) are selected for cross-linking with CNVK chains (grey filled circles) carrying a reporter fluorophore (orange star). Figure 8B shows an overlay of bright field and green channel images showing a mixture of GFP-transfected and untransfected cells. Multiple regions of interest (outlined in yellow, blue, green, red) selected for cross-linking are drawn around cells exhibiting GFP signal. FIG. 8C shows fluorescence images of cells after cross-linking. Nuclear staining (blue), GFP (green), and fluorescent CNVK chains (yellow) are superimposed. See description of Figure 8A. See description of Figure 8A. Figures 9A-9D show the sequencing results. Utilizing a variation of strategy 2 with UMIs on both ends of the amplicon, patterned irradiation was used on fixed HeLa cells to sequentially bar code three distinct spatially separated regions. bottom. Figure 9A demonstrates that six separate probe sequences, two targeting ribosomal RNA and four targeting Xist RNA, bound to their target RNA sequences using FISH. This was followed by repeated barcoding, binding of barcode-containing primers, recording synthesis, and amplification. Amplicons were prepared using the Collibri sequencing prep kit for next generation sequencing (HiSeq). Figures 9B-9C show that a high percentage of reads of the expected format were recovered after alignment. FIG. 9B discloses SEQ ID NOs: 15-16, respectively, in order of appearance. FIG. 9D shows the read distribution for most of the data , shown per probe-region pair . See description of Figure 9A-1. See description of Figure 9A-1. See description of Figure 9A-1. See description of Figure 9A-1. FIG. 10 demonstrates targeted and non-targeted approaches to barcoding. Any type of nucleic acid may be barcoded. These nucleic acids typically associate with, bind to, or hybridize to biomolecules that are localized in situ. Specific biomolecules can be targeted or affinity-based approaches, e.g., FISH for DNA/RNA targets, IF for protein targets (e.g., via nucleic acid-conjugated antibodies or Nanobodies), or conjugation with nucleic acids. Targeting can be through any other affinity-based reagent that can be gated or otherwise associated. A non-targeted approach may alternatively be utilized in which nucleic acids are localized or generated in a non-targeted manner . For example, cDNA copies produced from reverse transcription of RNA, or pre-existing RNA or DNA, or modified backbone sequences, or by the action of polymerases, ligases, restriction enzymes, nucleases, telomerase, terminal transferases, recombinases, or transposases. Other reaction products generated in situ can be barcoded, such as products of proximity ligation assays, primer exchange reactions, autocyclic proximity recordings or tagmentation. Figures 11A-11B show the assembly of barcodes to cells or other regions of interest. (FIG. 11A) Repeated formation of concatemers on nucleic acids (e.g., cDNA sequences) localized in situ results in the formation of barcodes (e.g., m-g-o-m-y-r-c) specific to reads from that cell. Although the orientation of the 3' barcoding of the cDNA is shown, 5' barcoding may be performed (see, eg, Figures 18 and 19). (FIG. 11B) Cross-junction synthesis and PCR are used to prepare recordings for sequencing. See description of FIG. 11A. FIG. 12 shows an application of the methods and compositions provided herein. See description for Figure 12-1. See description for Figure 12-1. Figure 13 shows the dissociative split-pool barcoding workflow. Division of cells or otherwise associated biomolecules (e.g., hydrogel pieces) into tubes, barcoding of nucleic acids, e.g., using light-induced concatemer formation as depicted elsewhere, and repeated repeated repooling. allows a unique barcode sequence to be associated with each distinct cell/component. Split-pool strategies have been used in the past for single-cell barcoding through multiple and expensive enzyme ligation steps, but using concatemer-based barcoding strategies, each barcoding step can be performed using expensive enzymes or enzymes. Cost is dramatically reduced as it can be performed without the need for other reagents. Sequences can be extracted using cross-junction synthesis and PCR of recordings as they are on the surface. See description for Figure 13-1. Figures 14A-14C illustrate certain aspects of spatial barcoding. (FIG. 14A) Barcodes are typically crosslinked through the use of CNVK modifications, and the crosslinks are activated with UV light. (FIG. 14B) By spatially addressing the UV light irradiation profile, barcodes can be cross-linked to Dock sequences only at desired positions, and after a stringent washing step (e.g., formamide-containing buffer), cross-linking Not all barcode strands can be washed away. (FIG. 14C) Repeated binding, cross-linking of a particular region, and stringent washing steps allow repeated construction of barcodes associated with that particular region. See description of Figure 14A. See description of Figure 14A. FIG. 15A shows linear barcoding of N regions (eg, N separate cells) is performed such that only one of the N barcodes is assigned to each location or locations of interest. I have demonstrated that. The sequencing results may then be bulk extracted together and the reads may be mapped back to their original corresponding positions based on the barcode sequences in the reads. Figure 15B demonstrates a method of combinatorial barcoding in which concatenated barcodes are repeatedly constructed (e.g. , see Figure 18). For example, for N rounds of M barcodes, M^N unique barcodes could be feasibly assigned. FIG. 16 shows one embodiment of a workflow for combining sample imaging data and RNA sequencing data. In general, additional imaging steps and other assays may be added before or after barcoding, and optionally an A-tailing step may be performed before or after barcoding. Different tailings (eg, T-tailing, C-tailing, G-tailing, or any other type of tailing with terminal transferase or other enzymes may be utilized) may alternatively be utilized. For the targeting approach, the workflow is very similar except that probes can already contain 5' and 3' tails, so both the RT and A tailing steps can be omitted. Any domain (eg, 1 letter, 2 letters, 3 letters or 4 letters) may be utilized for the 3' tail sequence. FIG. 17 shows experimental verification of UV power and irradiation conditions. A series of experiments to optimize UV power and illumination conditions for barcoding FISH probes bound to rRNA transcripts in HeLa cells. A checkerboard pattern was rastered across the wells with each distinct area to test different UV power and irradiation time conditions. Figure 18 shows a chain diagram of a 5' light-induced barcoding strategy with a UMI on the cross-junction synthetic primer. A primer with an overhanging 5' domain (eg, with random N bases at the end) is localized to RNA (eg, mRNA, non-coding RNA) to generate a cDNA sequence. The cDNA sequence may then have bases added on the 3' end, such as a polyA tail, using terminal transferase and dATP. Combinatorial barcodes are then assembled directly onto the 5′ overhang of the cDNA or other in situ localized sequences (either before or after barcoding) through steps of binding, UV cross-linking and washing. A tailing step may be included). Optionally, RNaseH replacement of barcodes from RNA may be performed prior to or concurrently with cross-junction synthesis. After cross-junction synthesis, a full transcript is formed via PCR amplification. See description for Figure 18-1. Figure 19 shows a strand diagram of a 5' light-induced barcode strategy with UMI on the barcode capping strand. A primer with an overhanging 5' domain (eg, with random N bases at the end) is localized to RNA (eg, mRNA, non-coding RNA) to generate a cDNA sequence. The cDNA sequence may then have bases added on the 3' end, such as a polyA tail, using terminal transferase and dATP. The combinatorial barcode is then assembled directly onto the 5' overhang of the cDNA or other in situ localized sequence through steps of binding, UV cross-linking and washing. Optionally, RNaseH replacement of barcodes from RNA may be performed prior to or concurrently with cross-junction synthesis. After cross-junction synthesis, a full transcript is formed via PCR amplification. See description for Figure 19-1. FIG. 20 shows experimental validation of primer sets for cDNA library construction. (Top) Table of primers and concentrations used for reverse transcription (RT). Well labels (A1-B4) match the orientation of the image shown at the bottom. Wells B1-B4 have primer combinations as well as negative controls that are not reverse transcribed. (Bottom) Image of cDNA library localization after reverse transcription using Cy5-labeled primers. A Cy3 CNVK barcode was then added, crosslinked in a checkerboard pattern using a DMD and a 10× objective, and imaged with Cy3. Figure 21 shows sequencing results for different RT primers. In situ reverse transcription in fixed HeLa cells was performed on the 5′ barcoding domain with NNNNNNN (7N, experiment A1), NNNNNGGG (5N and 3G, experiment A2) or NNNNNCCC (5N and 3C, experiment A3) on the 3′ end. was performed with different primers containing After barcoding, cross-junction synthesis and PCR according to the strategy depicted in Figure 18, PCR amplicons were purified with Ampure XP beads and sent for sequencing (250bp paired-end). Examples of some expected read results are shown for each of these primers, with the highlighted cDNA sequence (blue) mapping to a known Homo sapiens sequence as expected. These data demonstrate the success of this general strategy, in which each primer can be used successfully to generate a transcriptome recording. Figure 21 discloses SEQ ID NOs: 17-28, respectively, in order of appearance. See description for Figure 21-1. Figure 22A shows the sequence structure for barcoding 5' sequences (eg, 5' tail above cDNA, FISH probes, etc.). Concatemers formed using the reverse (Rev) primer capping strand, zero or more barcode strands, and cDNA, FISH, or other probe sequences with poly A tails were combined with the forward (For) primer and poly T3. Cross-junction synthesis primers containing the 'end can be used to effectively copy to form a PCR amplifiable record that can be sequenced. In this case, two different orientations of the barcode sequences (W/X and Y/Z domains) are utilized, although separate barcode sequences may be utilized as well. The strands may or may not be purified and may contain additional bases (eg, T-linkers, fluorophores, modifications to prevent extension or degradation) on the 3' or 5' ends. FIG. 22B shows certain embodiments of the binding domain barcode sequences used for demonstration in the next few figures , colored according to their domains . Any number of barcode strands with different (barcode) domain sequences may be utilized for barcoding. Figure 22B discloses SEQ ID NOs: 11-13, 11, 14, 12-14, and 13-14, respectively, in order of appearance. Figure 22C shows the complete sequence information for the experiments reported in all subsequent figures. PCR primer sequences are based on the Smart Seq3 protocol. All other sequences, and in particular those for barcoding, have been specifically and experimentally designed for this barcoding application after modeling and extensive testing of dozens of cross-junction synthesis reactions. See also Tables 1-3 in the Examples. FIG. 22C discloses SEQ ID NOs: 1-9, 29-31, and 10, respectively, in order of appearance. See description of FIG. 22A. See description of FIG. 22A. Figures 23A-23E show verification of repeated barcode assembly on streptavidin-coated surfaces (glass slides). FIG. 23A shows a schematic representation of repetitive barcode assembly of fluorescently labeled DNA barcode strands followed by cross-junction synthesis and PCR. FIG. 23B shows a schematic representation of concatenated barcodes with 2-7 junctions, each containing 1-6 barcodes. Figure 23C shows the expected DNA barcode length distribution in separate wells (top). The upper left well in the 8-well chamber contains a DNA barcode of length 6 and displays the highest amount of fluorescent signal. followed by 5 and 4 and so on. Scan of 8-well chamber in Cy3 fluorescence channel (bottom). Figure 23D shows the full sequence design of the 7-junction concatemer and amplicon based on the sequences presented in Figures 22A-22C. Figure 23D discloses SEQ ID NOs: 1, 8, 7, 6, 5, 4, 3, 10, 9, 30, 29, and 32, respectively, in order of appearance. FIG. 23E shows that amplicons from the top left well (6 junctions) were sequenced (250 bp paired-end sequencing) after extraction, PCR, and purification using a MinElute PCR purification column. Examples of sequencing results are shown for both full-length (reads containing 6 barcodes) and truncated reads (eg, containing 2 or 4 barcodes). Due to some inefficiencies in the concatemer formation process, truncated reads are expected in addition to full-length reads. Figure 23E discloses SEQ ID NOs: 33-39, respectively, in order of appearance. See description of FIG. 23A. See description of FIG. 23A. See description of FIG. 23A. See description of FIG. 23A. See description of FIG. 23A. See description of FIG. 23A. FIG. 24 shows sequencing results for several different fixation, permeabilization, RT and barcoding conditions following the strategy depicted in FIG. (Top) Shown are several sequences consistent with the expected sequence format after two rounds of barcoding, acquired for each of several fixation/permeabilization conditions (experiments B1-B8). These sequences show the expected barcode sequences in each case, as well as examples of the different UMIs and resulting sequence lengths. (Bottom) Several variations to the RT step were tested along with several controls, keeping fixation and permeabilization constant. For each of experiments C1-C4, one barcode was first introduced that was not crosslinked prior to stringent washing (exchange 1), then a second barcode that was UV crosslinked and should appear in the sequencing reads. Introduced barcode (exchange 2). As expected, cross-linked correct barcodes appeared in the majority of reads (>1,500 out of 2,000 reads examined) and incorrect ( Uncrosslinked exchange 1 barcode) barcodes appeared very infrequently (as low as 0 in 2,000 reads). In all conditions (experiments B2-B8, C1-C4) except the no reverse transcriptase (RT) control (experiment B1), the highlighted cDNA sequences (blue) map to known Homo sapiens sequences. . Exceptions: some conditions with A-tailing are performed after barcoding as indicated in the figure, all conditions with RNaseH treatment are combined with cross-junction synthesis incubation. Figure 24 discloses SEQ ID NOs: 40-75, respectively, in order of appearance. See description for Figure 24-1. See description for Figure 24-1. See description for Figure 24-1. See description for Figure 24-1. Figures 25A-25D demonstrate the imaging and gel results of experiments B1-B8 and C1-C4. FIG. 25A shows the imaging results of experiments B1-B8, showing distinct fluorescence morphologies after reverse transcription (RT) using fluorophore (Alexa 488) labeled RT primers. As expected, the fluorescent signal from the localized primers dropped significantly after displacement, indicating that they were successfully displaced during the combinatorial RNaseH and cross-junction synthesis steps. Figure 25B shows that the signal was much higher for the control condition containing RNase A but no RNaseOUT during RT, with lower contrast visualization revealing a strongly suspected nucleolar signal. . FIG. 25C shows the imaging results of experiments C1, C3 and C4. FIG. 25D shows the length of recordings generated after PCR amplification (1% agarose E-gel with Sybr Gold) , showing gel results for all conditions . Typical lengths recovered for those containing reverse transcription but no RNase A range from about 150 bp to 1300 bp. See description of Figure 25A. See description of Figure 25A. See description of Figure 25A. See description of Figure 25A. FIG. 26 shows transcriptome mapping results. Transcriptome mapping was performed using the STAR aligner on the sequencing results. (Left) An example output log file is shown on the left for the mapping results of 1,024 transcripts identified as having the expected sequence format for experiment B7. 40.5% of the reads mapped uniquely, while 49% mapped to multiple loci and 9.5% were too short to map. (Right) Gene mapping results are sorted by the frequency of mapped transcripts, delineating the top of the list. The most uniquely mapped genes corresponded to mitochondrial rRNA. See description for Figure 26-1. Figure 27 shows automatic barcode assignment and repetitive barcodes on a surface. An example workflow that can transform a list of barcodes (BC1, BC2, BC3, etc.) into a series of photomasks (middle panel) where each region of interest (white squares, middle panel) is assigned a unique barcode. Imaged after a series of 6 barcoding steps with fluorescent DNA strands, uniquely tagged and barcoded into an array of 112 regions of interest (right panel). Figures 28A-28G show automated barcoding of biomolecular samples. FIG. 28A shows a workflow by which cell collections can be detected with a computer algorithm and selectively targeted for barcode delivery, resulting in each cell having a unique barcode assignment. Figure 28B shows an image of cells with fluorescent DNA primers targeting RNA. Figure 28E shows images of cells after 6 rounds of barcoding with fluorescent DNA barcodes (green) using masks from panels (Figures 28C, 28F). Figures 28C and 28F show overlays of detected cell masks (white outlines). Figures 28D and 28G show enlarged images of the square outlines from (Figure 28C) and (Figure 28F), respectively. See description for Figure 28-1.

Exemplary modified nucleobases include inosine, xanthine, hypoxanthine, nubularine, isoguanosine , tubercidin , and substituted or modified analogs of adenine, guanine, cytosine and uracil. , e.g., 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil , cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-aminoallyluracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and others 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanines, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substitutions of Purines (including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine), dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurines, 5-alkyluracils, 7-alkylguanines, 5-alkyl Cytosine, 7-deazaadenine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole , 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2 -Thiouracil, 3-(3-amino-3-carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N ⁴ -acetylcytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio Including, but not limited to, -N6-isopentenyl adenine, N-methylguanine, or O-alkylated bases. Further purines and pyrimidines are disclosed in U.S. Pat. No. 3,687,808, Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990. , and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613.

In some embodiments, the modified nucleobases are inosine, xanthine, hypoxanthine, nuvalarine, isoguanosine , tubercidin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-( Amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(methylthio) ^-N6- (isopentenyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7 -(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8- (thioalkyl)adenine, 8-(thiol)adenine, ^N6- (isopentyl)adenine, ^N6- (methyl)adenine, ^N6 , ^N6- (dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl) )guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl) Guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, N-(methyl)guanine , 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5 -(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, ^N4- (acetyl)cytosine , 3-(3-amino-3-carboxypropyl)uracil, 5-ethynyl-2'-deoxyuridine, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylamino Methyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)uracil -2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil , 5-(allylamino)uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(1,3-diazole-1-alkyl)uracil, 5- (Cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonyl) methyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, Dihydrouracil, N ³ -(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 5-( Alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-( Thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil , 1-substituted pseudouracils, 1-substituted 2- (thio)-pseudouracils, 1-substituted 4-(thio)pseudouracils, 1-substituted 2,4-(dithio)pseudouracils, 1-(aminocarbonylethylenyl )-Pseudouracil, 1-(aminocarbonylethylenyl) -2- (thio)-pseudouracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethylenyl)-2 ,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminoalkylamino-carbonylethylenyl) -2- (thio)-pseudouracil, 1-(aminoalkylamino carbonylethylenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazine -1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1 -yl, 1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-substituted 1,3-(Diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7 -substituted 1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7 -(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3- (Aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1-( Aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-(guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazine-1- yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1, 3-(Diaza)-2-(oxo)-phenothiazin-1-yl, 7-(guanidinium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazine-1- yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nuvalarine, tubercidin, isoguanosine , inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl , Nitroimidazolyl, Nitropyrazolyl, Nitrobenzimidazolyl, Nitroindazolyl, Aminoindolyl, Pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7 -(propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl, 2,4,5- Rimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6- (methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2 ,6-(diamino)purines, 5-substituted pyrimidines, ^N2 -substituted purines, ^N6 -substituted purines, ^O6 -substituted purines, substituted 1,2,4-triazoles, and any O-alkylation thereof or It can be selected from the group consisting of N-alkylated derivatives.

In some embodiments of the various aspects described herein, the nucleic acid strands described herein use linkers or spacers, e.g., at internal positions at the 3' and/or 5' ends. can be modified above. While not wishing to be bound by theory, linkers or spacers can be used to link nucleic acid strands to moieties such as solid supports or labels. In some aspects, the linker or spacer is a photocleavable linker, hydrolyzable linker, redox cleavable linker, phosphate cleavable linker, acid cleavable linker, ester cleavable linker, peptide cleavable linker , and any combination thereof. In some embodiments, the cleavable linker is a disulfide bond, a tetrazine-trans-cyclooctene group, a sulfhydryl group, a nitrobenzyl group, a nitroindoline group, a bromohydroxycoumarin group, a bromohydroxyquinoline group, a hydroxy It can contain phenacyl groups, dimethoxybenzoin groups, or any combination thereof.

1,8-ANS; 4-methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); Naphthofluorescein (pH 10); 5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-Carboxyfluorescein); 5-Hydroxytryptamine (HAT); 5-ROX (Carboxy-X-rhodamine); TAMRA (5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 4-methylcoumarin; 9-amino-6-chloro-2-methoxyacridine; ABQ; acid fuchsine; ACMA (9-amino-6-chloro-2-methoxyacridine); acridine orange; acridine red; Acriflavin Feulgen SITSA; Aequorin (photoprotein); Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; APC); AMC, AMCA-S; AMCA (aminomethylcoumarin); AMCA-X; aminoactinomycin D; aminocoumarin ; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH) berberine sulfate; beta-lactamase; BFP blueshift GFP (Y66H); BG-647; Bodypy 515; Bodypy 493/503; Bodypy 500/510; Bodypy 505/515; Bodypy 530/550; Bodypy 542/563; Bodipy 650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; BO-PRO™-1; BO-PRO™-3; Brilliant Sulfoflavin FF; Calcein; Calcein Blue; Calcium Crimson™; calcium green-1 Ca2 ⁺ dye; calcium green-2 Ca2 ⁺ ; calcium green-5N Ca2 ⁺ ; calcium green-C18 Ca2 ⁺ ; calcium orange; 5-ROX); Cascade Blue™; Cascade Yellow; Catecholamines; CFDA; CFP-Cyan Fluorescent Protein; Chlorophyll; Coelenterazine hcp; Coelenterazine ip; Coelenterazine O; Coumarin Phalloidin; CPM Methylcoumarin; CTC; Cyclic AMP fluorosensor (FiCRhR); d2; Dabsyl; Dansyl; Dansylamine; Dansylcadaverine; Dansyl chloride; Dansyl DHPE; 2; dapoxyl 3; DCFDA; DCFH (dichlorodihydrofur olecein diacetate); DDAO; DHR ( dihydrorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS ( non -ratio); DHR); DiO (DiOC18(3)); DiR; DiR (DiIC18(7)); Dopamine; DsRed; DTAF; Erythrosine; Erythrosine ITC; Ethidium Homodimer-1 (EthD-1); Euchrysin; Europium(III) Chloride; Fluor-Emerald; Fluoro-Gold (hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; ) (high pH); Fura-2, high calcium; Fura-2, low calcium; Genacryl brilliant red B; Genacryl brilliant yellow 10GF; Genacryl pink 3G; Genacryl yellow 5GF; (rsGFP); GFP wild-type, non-UV-excited (wtGFP); GFP wild-type, UV-excited (wtGFP); GFPuv; Hydroxystilbamidine (Fluorogold); Hydroxytryptamine; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; Laurodan; LDS 751; Leucophor PAF; Leucophor SF; Leucophore WS; Lissamine Rhodamine; Lissamine Rhodamine B; Green; Magnesium Orange; Malachite Green; Marina Blue; Maxiron Brilliant Flavin 10 GFF; Maxiron Brilliant Flavin Mitotracker Green FM; Mitotracker Orange; Mithramycin; Monobromobimane; Monobromobimane (mBBr-GSH); NBD; NBD Amine ; Nile Red; Nitrobenzoxadiazole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; Photoresist; Phycoerythrin B [PE]; PKH26; PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; rhodamine BB; rhodamine BG; rhodamine green; rhodamine faricidin; rhodamine phalloidin; S65T; Sapphire GFP; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; SITS (Primulin); SITS (stilbeneisothiosulfonic acid); SPQ (6-Methoxy-N-(3-sulfopropyl)-quinolinium); Stilbene; Sulforhodamine B can C sulforhodamine G extra; tetracycline; tetramethylrhodamine; Texas Red™; Texas Red-X™ conjugate; Thiolyte; Thiosol Orange; Tinopol CBS (CalcoFluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TRITC (tetramethylrhodamine isothiocyanate); True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; Many suitable forms of these fluorescent compounds are available and can be used.

Other exemplary detectable labels include luminescent and bioluminescent markers (e.g., biotin, luciferase (e.g., those of bacteria, fireflies, click beetles, etc.), luciferin and aequorin), radioactive labels (e.g., 3H, 125I, 35S, 14C or 32P), enzymes (e.g. galactosidases, glucorinidases, phosphatases (e.g. alkaline phosphatase), peroxidases (e.g. horseradish peroxidase), and cholinesterase), and colorimetric labels such as colloidal gold or colored glass. or plastic (eg, polystyrene, polypropylene and latex) beads. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; each of which is incorporated herein by reference in its entirety.

Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with an enzyme substrate and detecting the reaction product produced by the action of the enzyme on the enzyme substrate; colorimetric labels are detected by visualizing the colored label. can be detected.

In some embodiments, two different domains can contain identical nucleotide sequences. In some aspects, a nucleic acid strand can include a restriction site. For example, restriction sites can be used within the joining region between the ligating barcode strands , and hairpins can be ligated to the cleaved ends to form the complete recording strand . Alternatively, the strands bridging the junction can be attached to the assembly and then ligated together.

In another aspect, the method comprises (a) hybridizing a target nucleic acid and a first nucleic acid, wherein (i) the first nucleic acid is oriented 5' to 3' in (1) optionally, a unique molecular identifier (UMI) sequence; (2) a first targeting domain, the first targeting domain being substantially complementary to the target nucleic acid; and (3) a first hybridization domain. (b) preparing concatemers by hybridizing n additional nucleic acids and photocrosslinking the additional nucleic acids with the first complex, wherein n is any is an integer from 1 to 100, and each additional nucleic acid comprises, in the 5' to 3' direction, (i) a first hybridization domain; (ii) a barcode domain; and (iii) a second hybridization domain. wherein the first hybridization domain of the nth nucleic acid is substantially complementary to the second hybridization domain of the (n−1)th nucleic acid, and the first hybridization domain of n=1 nucleic acids; are substantially complementary to the first hybridization domain of the first nucleic acid, and at least one of the first or second hybridization domains of each nucleic acid comprises a photoreactive element (c) hybridizing the first cap nucleic acid and the concatemer, thereby forming a capped concatemer, wherein the first cap nucleic acid undergoes (i) the first cap hybridization a first cap hybridization domain substantially complementary to a second hybridization domain of the nth nucleic acid; and (ii) a second cap hybridization domain; d) hybridizing a second cap nucleic acid to the capped concatemer, thereby forming a concatemer-primer complex, wherein the second cap nucleic acid is oriented 5′ to 3′, (i (ii) optionally a unique molecular identifier (UMI) sequence; and (iii) a hybridization domain substantially complementary to the second cap hybridization domain of the first cap nucleic acid. a hybridization domain, a second hybridization domain of the first cap nucleic acid and a hybridization domain of the second cap nucleic acid and (e) detecting the concatemer-primer complex or synthesizing the recording nucleic acid from the concatemer-primer complex and detecting the recording nucleic acid. .

Strategy 3: Light-Guided Barcoding Using Concatemer Assembly:
A third strategy also uses only one wavelength of light (approximately 365 nm) to cross-link CNVK-containing sequences to semi- or fully complementary sequences. This strategy utilizes multiple rounds of cross-linking directed to the same region or sequence such that multiple-stranded complexes (concatemers) are assembled (see Figures 3A-3C). Cross-junction synthesis can then be used to copy strands of barcode sequences on concatemers into sequenceable recording strands.

Figures 9A-9D show the sequencing results. Utilizing a variation of strategy 2 with UMIs on both ends of the amplicon, patterned irradiation was used on fixed HeLa cells to sequentially bar code three distinct spatially separated regions. bottom. Figure 9A demonstrates that six separate probe sequences, two targeting ribosomal RNA and four targeting Xist RNA, bound to their target RNA sequences using FISH. This was followed by repeated barcoding, binding of barcode-containing primers, recording synthesis, and amplification. Amplicons were prepared using the Collibri sequencing prep kit for next generation sequencing (HiSeq). Figures 9B-9C show that a high percentage of reads of the expected format were recovered after alignment. FIG. 9D shows the read distribution for most of the data , shown per probe-region pair .

(Table 2) Specific structures of sequences with d0 and d1 binding domains (see also Figure 22B). A specific set of experimentally validated barcoding binding domains is described (d0 = (binding domain W) listed in Table 1 and d1 = (binding domain Y) from Table 1). The binding domain is sufficient so that uncrosslinked barcode strands can be washed away without destroying the underlying affinity or binding of the docking sequences (e.g., cDNA sequences or localized FISH or targeting probes). It must be designed short.

Claims (22)

a. in the 3' to 5' direction, or in the 5' to 3' direction,
i. optionally, a unique molecular identifier (UMI) sequence;
ii. a first targeting domain; and
iii. a first nucleic acid comprising a first hybridization domain;
b. in the 5' to 3' direction, or in the 3' to 5' direction,
i. a first barcode domain; and
ii. a second hybridization domain, the second hybridization domain being substantially complementary to the first hybridization domain of the first nucleic acid; A code composition,
The barcode composition, wherein at least one of the first or second hybridization domain comprises a photoreactive element. 2. The barcode composition of claim 1, wherein the second nucleic acid further comprises a unique molecular identifier sequence at one of the 3' end or the 5' end. The barcode of claim 1 or 2, further comprising a third nucleic acid comprising a second barcode domain, said second barcode domain being substantially complementary to said first barcode domain. Composition. 4. The barcode composition of claim 3 , wherein the third nucleic acid further comprises a unique molecular identifier sequence at the 3' or 5' end. further comprising n additional nucleic acids;
n is an integer from 1 to 100,
each additional nucleic acid in the 3' to 5' direction, or in the 5' to 3' direction,
i. first hybridization domain;
ii. barcode domain; and
iii. a second hybridization domain;
the first hybridization domain of the nth nucleic acid is substantially complementary to the second hybridization domain of the (n-1)th nucleic acid;
the first hybridization domain of n=1 nucleic acids is substantially complementary to a third hybridization domain, and at least one of the first or second hybridization domain of each nucleic acid is 2. The barcode composition of claim 1 , comprising a photoreactive element. in the 3' to 5' direction, or in the 5' to 3' direction,
i. a first cap hybridization domain, the first cap hybridization domain being substantially complementary to a second hybridization domain of the nth nucleic acid when n is 1 or more; or 0, the cap hybridization domain is substantially complementary to a third hybridization domain; and
ii. further comprising a first cap nucleic acid strand comprising a second cap hybridization domain;
6. The barcode composition of claim 5 , wherein the first cap hybridization domain optionally includes a photoreactive element. in the 3' to 5' direction, or in the 5' to 3' direction,
i. Primer sequence domain;
ii. Optionally, a unique molecular identifier (UMI) sequence; and
iii. further comprising a second cap nucleic acid strand comprising a hybridization domain, the hybridization domain being substantially complementary to the second cap hybridization domain of the first cap nucleic acid;
7. The barcode composition of claim 6 , wherein at least one of the second cap hybridization domain of the first cap nucleic acid strand and the hybridization domain of the second cap nucleic acid strand comprises a photoreactive element. . 3. The barcode composition according to claim 1, wherein the first nucleic acid further includes a primer sequence at its 5' end. 3. The barcode composition of claim 1 or 2 , wherein the first targeting domain of the first nucleic acid is substantially complementary to a target nucleic acid. The target nucleic acid is conjugated to a target binding substance, or the target nucleic acid is conjugated to a target molecule, or the target nucleic acid is contained within a target molecule, or A nucleic acid is expressed by a target cell, or the target nucleic acid is directly attached to a target molecule or cell, or by chemical cross-linking, genetic encoding, viral transduction, transfection, conjugation, cell fusion, 10. A barcode composition according to claim 9 , which is presented indirectly via cellular uptake, hybridization, DNA binding proteins or adapter molecules, such as target binding ligands. The target binding substance may include amino acids, peptides, proteins, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, lipopolysaccharides, lectins, nucleosides, nucleotides, nucleic acids, vitamins, steroids, hormones, cofactors, receptors, and receptors. 11. The barcode composition of claim 10 , wherein the target binding substance is an antibody or antigen-binding fragment thereof. 3. A barcode composition according to claim 1 or 2 , wherein the UMI of a nucleic acid is incorporated into one of the other domains of the same nucleic acid. 3. The barcode composition of claim 1 or 2 , wherein at least one of the nucleic acids comprises a cleavable spacer. detectable label ;
Strand displacement polymerase;
Buffers or salts for nucleic acid synthesis;
natural or synthetic nucleotide triphosphates or deoxynucleotide triphosphates;
target element;
PCR primer; or
a light source, optionally the light source being a UV light source
3. The barcode composition according to claim 1 or 2 , further comprising: The detectable label is
contained within one of said nucleic acids, or
Fluorescent molecules, nanoparticles, stable isotopes, radioactive isotopes, nucleotide chromophores, enzymes, enzyme substrates, chemiluminescent and bioluminescent moieties, echogenic materials, nonmetallic isotopes, optical reporters, paramagnetic metal ions, and selected from the group consisting of magnetic metals, optionally said detectable label being a fluorophore;
15. The barcode composition according to claim 14 . 3. A barcode composition according to claim 1 or 2 , wherein the photoreactive element is a photoreactive nucleotide, and optionally the photoreactive nucleotide is a CNVK or CNVD bridging base. 2. The barcode composition of claim 1 in the form of a kit. A method of detecting target mRNA, the method comprising the steps of:
a. A step of hybridizing target mRNA (first nucleic acid) and second nucleic acid,
i. the mRNA comprises a first hybridization domain comprising a polyA sequence;
ii. the second nucleic acid in the 5' to 3' direction,
1. a second hybridization domain, the second hybridization domain being substantially complementary to the first hybridization domain and comprising a photoreactive element; and
2. a step containing a first barcode domain;
b. photocrosslinking the mRNA and the second nucleic acid, thereby forming a probe-primer complex;
c. synthesizing a recording nucleic acid from the probe -primer complex; and
d. Detecting the recorded nucleic acid. 10. The barcode composition of claim 9, wherein the first targeting domain of the first nucleic acid is substantially complementary to RNA or an RNA transcript. Claim 1 or 2, wherein the second nucleic acid further comprises a third hybridization domain at either the 3' end or the 5' end, said third hybridization domain optionally comprising a photoreactive element. The barcode composition described. 3. The barcode composition of claim 1 or 2, wherein the first nucleic acid further comprises a 3' tail sequence, the tail sequence comprising an A-tailing, a T-tailing, a C-tailing, a G-tailing, or any combination thereof. thing. 22. The barcode composition of claim 21, wherein the tail sequence comprises a polyA sequence.